The proposed research is intended to discover novel anti-cancer agents that inhibit expression of thymidylate synthase (TS), the sole intracellular de novo source of thymidylate for DNA biosynthesis. Specifically, we want to synthesize and identify compounds that selectively bind and stabilize a structured regulatory element in the 5' nontranslated region (NTR) of the TS mRNA which contains the translation start codon sequestered in an RNA hairpin element. By preventing access of the cellular translation machinery to the initiation site, TS mRNA-binding compounds would mimic the action of TS protein which acts as a ligand and repressor of its own mRNA. In preliminary experiments we have validated the TS mRNA as a target for translation inhibition via stabilization of the mRNA secondary structure. In the proposed research we will establish an iterative process aiming at the discovery of lead compounds for the development of novel cancer therapeutics for use in combination with existing anti-cancer drugs that target the enzyme but suffer from resistance development via upregulation of TS expression. The specific aims of this project are to: 1) design and synthesize novel RNA-biased ligands based on two classes of heterocycles privileged for RNA binding; 2) design and validate model oligonucleotides representing the regulatory RNA element in the 5'-NTR of TS mRNA for ligand binding studies; 3) determine the three- dimensional structure of the regulatory RNA element in the TS 5'-NTR by X-ray crystallography; 4) develop RNA affinity and target specificity assays for the TS mRNA and identify small molecule binders among the newly synthesized RNA-biased ligands and known RNA-binding natural products; 5) evaluate TS mRNA binders for specific target recognition in binding and in vitro translation assays; 6) test TS-specific translation inhibitors in human cell lines to determine membrane permeability, interference with TS expression and antiproliferative activity; 7) determine the three-dimensional structure of TS mRNA-ligand complexes by X-ray crystallography; 8) design modified ligands with potentially improved binding affinity by using biological activity data and structural information (structure activity relationships, SAR).